The distribution and characterization of met-5-enkephalin-arg-6-gly-7-leu-8-(met-enk-arg-gly-leu) and peptide F in the rat gastrointestinal (GI) tract was studied by immunohistochemical techniques. Antisera were found to be specific for the synthetic antigens. Met-enk-arg-gly-leu and peptide F-like immunoreactivities were found in neuronal structures in all regions of the rat GI tract. Immunoreactive somata were primarily located in the myenteric plexus; immunoreactive processes were mostly present in the myenteric plexus and the circular muscle layer. By comparing the distribution of these two peptides and other enkephalin-containing peptides (met-5-enkephalin, leu-5-enkephalin and met-5-enkephalin-arg-6-phe-7), we found that their distribution is similar. It is most likely that these five peptides coexist in the same neurons of the rat GI tract. Our results suggest that these opioid peptides may play a role in the regulation of GI functions.